Regulatory Effcts Of Nonclassical Benzodiazepine Binding Sites On GABA_A Receptors

Benzodiazepines（BZDs）are classical sedative-hypnotics.Diazepam（DZP）,midazolam（MDZ）and zolpidem（ZOL）are commonly-used BZDs in clinic.BZDs target toγ-aminobutyric acid type A receptor（GABA_AR）to induce various effects,including anti-anxiety,sedation,muscle relaxation and anesthesia.Due to their versatile pharmacological actions,when one of the actions is used as therapeutic effect,BZDs often brought various side effects.Flumazenil（FLU）,a well-defined BZD antagonist,is considered to selectively block various central effects of BZDs both in experimental animals and in humans.However,accumulating clinical evidence shows that flumazenil was inefficient in antagonizing BZD effects under certain circumstances.It is generally believed that BZDs bind to a site（the classical binding site）,which is located at the extracelluar interface ofαandγsubunits of GABA_Areceptor.However,several non-classical benzodiazepine binding sites have been subsequently revealed on certain GABA_A receptor subtypes,and BZDs effects via non-classical binding sites were insensitive to FLU.Various binding sites determine the versatile BZDs actions.Non-classical binding sites may,to some extent,compensate for the failure antagonism of FLU on certain BZDs.So far three non-classical binding sites,which are located at transmembrane domain,α+/β-interface orβ-/γ+interface,have been reported on some GABA_A receptors.It has been postulated that the classical site was mainly involved in the sedative and anti-anxiety effects induced by relative low-dose BZDs,while non-classical binding sites contribute to the anesthetic effect induced by high-dose BZDs.Our previous study showed FLU-insensitive DZP effecs on zebrafish spontaneous activity model.The present study further explore regulatory effects of non-classical BZD binding sites in various recombinant and neuronal GABA_A receptors,exploring the role of non-classical binding sites in anesthesia induced by BZDs.Objective:The high-concentration effects of three benzodiazepines（diazepam,midazolam and zolpidem）and their sensitivity to flumazenil were compared on the recombinantα1β2 GABAA receptor using the whole-cell patch-clamp electrophysiology technique.To explore the contribution of receptor subunits in forming the non-classical benzodiazepine binding sites,benzodiazepine effects on various GABA_A receptor subtypes（α₂β₂,α₃β₂,α₄β₂,α₅β₂,α₁β₃）were investigated.In addition,effects of diazepam were tested onα₁β₂γ₂ receptor.Cortical and hippocampal neurons were used to confirm the existence of non-classical benzodiazepine effects in native GABA_Areceptors.Finally,the role of non-classical binding sites in BZDs mediated anesthesia effect was studied in mice.Methods:1. Effects of BZDs on recombiant GABA_A receptorαβ receptors（composed of onlyαandβsubunits）do not contain the classical BZD sites（α+/γ-）,this type of receptors were used as a model to study non-classical binding sites.In this study,sixαβreceptors were constructed:c DNAs ofα1,α2,α3,α4andα5 subunits were cloned into vector pc DNA3.1（+）and that ofβ2 andβ3 subunits into vector p IRES2（EGFP）,which allowed the expression of both theβ2 orβ3 subunit and green fluorescent protein from an internal ribosomal entry site（IRES）.HEK293cells were transfected with combinations of differentαandβ/EGFP subunits c DNAs to recombineαβreceptors.Compared withαβreceptors,it is more difficult to expressα₁β₂γ₂ receptor in HEK293 cells.GABA_A receptor associated protein（AP）contributes to the correct assembly of different subunits,soα1,β₂,γ₂ and AP were cotransfected into HEK293 cells to recombineα₁β₂γ₂receptor.The c DNA fragments AP were cloned into vector pc DNA3.1.The green fluorescence of EGFP is used to indicate the expression of the recombinant receptor.EGFP-positive cells were used for electrophysiological recording 48 h after transfection.Whole-cell patch clamp technique was adopted to detect the allosteric effects of BZDs on different GABA_Areceptor subtypes and its sensitivity to FLU.2.Effects of DZP on hippocampal and corticalHippocampal and cortical neurons were isolated and cultured for 7-14 days.Immunofluorescence was used to identify neurons.The effects of DZP on neurons and its sensitivity to FLU were studied using whole-cell patch clamp technique.3.The role of nonclassical binding sites in BZD-induced anesthesiaAnesthesia induced by BZDs was measured using a mice loss of righting reflex（LORR）model.To study the preventive and therapeutical effects of FLU on diazepam-induced anesthesia,FLU was injected immediately before or 20 min after diazepam injection.The effects of GABA_A receptor blocker bicuculline（BIC）and pentylenetetrazol（PTZ）on DZP-induced LORR were tested to confirm whether the anesthetic effects of DZP were mediated through GABA_A receptor.The role of PKA in DZP-induced LORR was studied to explore whether high dose DZP produced its effects through non-GABAergic system.Effects of FLU on midazolam（MDZ）and zolpidem（ZOL）were also investigated.Results:1. The effects of DZP on recombinant GABA_A receptors1.1 GABA concentration-dependent activation of recombinantαβreceptorsCells showed green fluorescence,which indicates successful expression,48h after transfection.GABA concentration-dependently activatedα₁β₂,α₂β₂,α₃β₂,α₄β₂,α₅β₂andα₁β₃ receptors with EC₅₀ values of 1.23 7.05,7.94,0.37,7.12 and 1.13μmol/L.Hill coefficient values were different among disrinct receptors.According to the EC₅₀values,the relative order of GABA potency onαβreceptors was:α₄β₂>α₁β₂≈α₁β₃>1.2 α₁β₂ receptor showed distinct sensitivities to different BZDsThe prototype l,4-benzodiazepine DZP（200μmol/L）elicited a marked modulatory（P<0.05,n=4）,and the potentiation was not affected by 200μmol/L FLU.Similar to DZP,MDZ（200μmol/L）,which owns the 1,2-annelated imidazo-benzodiazepine structure,also significantly potentiated GABA current（171.8±13.4%,P<0.05,n=4）in a FLU-insensitive manner.However,the imidazopyridine ZOL（200μmol/L）failed to effectively modulate the receptor.1.3 The contribution ofαandβsubunits to the sensitivity ofαβreceptor to DZPTo clarify the contribution of specificαsubunit in mediating BZD effect,effects of DZP onα₂β₂,α₃β₂,α₄β₂ andα₅β₂ receptors were tested.In concordance with the results onα1β2 receptor,DZP（200μmol/L）significantly potentiated the EC_3-10GABA currents uponα₂β₂（213.3±26.7%,P<0.05,n=3,）,α3β2（183.2±21.1%,P<0.05,n=5）andα₅β₂（316.2±71.5%,P<0.01,n=6,）receptors,the potentiation was not significantly reduced by flumazenil（200μmol/L）.However,theα₄β₂ receptor could not be effectively modulated by 200μmol/L diazepam.The α₁β₃ receptor was also tested in comparison toα₁β₂ receptor,in order to study whether the diazepam effect depend onβsubunit present in the receptors.Unlike theβ2-containing receptors,α1β3receptor was insensitive to diazepam in the present study.1.4 Effects of DZP on recombinantα₁β₂γ₂ receptorEC_3-10GABA current were not potentiated by DZP（200μmol/L）onα₁β₂γ₂receptor.Further study showed that,low concentration DZP（0.1,10,100μmol/L）elicited a marked modulatory effect onα₁β₂γ₂ receptor,potentiating the EC_3-10 GABA currents to 218.6%,203.3%and 141.1%respectively.However,GABA current in high-concentration DZP（500μmol/L）treated group was similar to that in the control group.These results indicated that lower concentration of DZP binded to the classical site to modulateα₁β₂γ₂ receptor,whereas DZP at higher concentration might bind to another site to antagonize the classical effects.2. Effects of DZP on neuronal GABA_A receptors2.1 GABA concentration-dependent effects on hippocampal and cortical neuronsHippocampal and cortical neurons were cultured for 7-14 days before electrophysiological experiments.GABA activated hippocampal and cortical neurons in a concentration-dependent manner,with EC₅₀ values of 5.1 and 7.51μmol/L respectively.2.2 Effects of DZP on some cortical neurons could not be fully antagonized by FLUNeurons showed distinct responses to DZP and FLU.In the 7 hippocampal neurons recorded,DZP（200μmol/L）potentiated GABA EC_3-10 currents to 239.4±17.4%（P<0.01,n=3）analyzed on（3/7）neurons,FLU（200μmol/L）completely antagonized the effects of diazepam（P<0.01）.However,the other（4/7）neurons were insensitive to 200μmol/L DZP.Similar to hippocampal neurons tested,（4/10）cortical neurons were modulated by 200μmol/L DZP（244.5±62.6%,P<0.05,n=4）and the DZP effects was completely antagonized by 200μmol/L FLU（P<0.05）,（4/10）cortical neurons were insensitive to DZP.However,FLU only partially antagonized DZP effects on（2/10）neurons.The potentiation of DZP（200μmol/L）on GABA EC_3-10 currents was 302%and 551%respectively,and FLU（200μmol/L）decreased the elicited current to 168%and 207%.3. The role of non-classical BZD binding sites in BZD-induced anesthesia3.1 Effects of FLU on LORR-induced by DZPDZP（10-100 mg/kg）dose-dependently increased the percentage of LORR in mice.An increase in DZP dose led to a decrease in the latency and an increase in the duration of LORR.DZP at dose of 50 mg/kg caused 100 percent of LORR.50 mg/kg DZP was used as the middle dose to study the effects of FLU on DZP-induced LORR.To analyze the sensitivity to FLU of potentially-different anesthetic states induced by different doses of DZP,DZP at a lower dose of 25 mg/kg and a higher dose of 100mg/kg were also studied in comparison with 50 mg/kg DZP.FLU pretreatment produced a dose-dependent trend in increasing the latency to LORR induced by 25 and 50 mg/kg DZP.The mice pretreated with 0.5 mg/kg FLU exhibited a significantly longer latency to LORR induced by 25 mg/kg DZP,compared with the vehicle-treated mice（P<0.001）.FLU at doses of 2 mg/kg（P<0.001）and 4mg/kg（P<0.05）prolonged the latency of LORR induced by 100 mg/kg DZP.The mice treated with a higher dose of 16 mg/kg FLU,however,exhibited similar latency to LORR induced by 100 mg/kg DZP,compared with the vehicle-treated mice.Unexpectedly,FLU pretreatment failed to reduce the duration of LORR in all DZP-injected mice.Moreover,1 mg/kg FLU significantly prolonged duration of 50mg/kg DZP-induced LORR in mice.The mice pretreated with FLU（1.0-16.0 mg/kg）exhibited a trend in increasing the duration of LORR induced by 100 mg/kg DZP,compared with the mice treated with vehicle,although the differences were not significant.FLU dose-dependently increase the latency to Re-LORR induced by 50Similar to the results in FLU pretreatment experiment,FLU post-treatment failed to reduce the duration of re-LORR induced by DZP.Moreover,the duration of 100 mg/kg DZP induced Re-LORR was significantly prolonged by FLU(F_{（6,65）}=2.496,P<0.05).3.2 Effects of GABA_A receptor blockers on DZP-induced LORREffects of GABA_A receotor antagonist BIC and PTZ on DZP were investigated to confirm whether LORR induced by diazepam was mediated via GABA_A receptors.BIC（0.25 mg/kg）pretreatment showed no significant effect on DZP-induced LORR.PTZ（20 mg/kg）Pretreatment significantly increased the latency（t=2.88,P=0.01）and reduced the duration（t=3.3,P<0.01）of LORR induced by DZP.Similarly,the mice post-treated with PTZ exhibited longer latency（t=2.75,P<0.05）and a shortening trend in the duration of DZP-induced re-LORR,compared with the vehicle-treated miceTo confirm whether DZP produced effects via non-GABAergic system,we further studied the role of PKA in DZP-induced LORR.H89（i.c.v.）,a PKA inhibitor,pretreatment produced no significant effects on latency of DZP-induced LORR,the LORR duration was prolonged by H89(F_{（3,38）}=2.99,P<0.05).However,8-Br-c AMP（PKA activator）showed no significant effects on LORR induced by DZP.3.3 Effects of FLU on BZD-induced LORR depends on BZD structureMDZ and ZOL dose dependently induced LORR in mice.MDZ and ZOL at doses of 100 and 50 mg/kg respectively caused 100%LORR.Then,these doses were chosen to study the effects of FLU on the LORR induced by MDZ and ZOL.Similar to DZP,MDZ（100 mg/kg）induced LORR could not be effectively antagonized by FLU.FLU produced no significant effects on percentage and latency of LORR induced by MDZ.Moreover,the LORR duration induced by MDZ was significantly prolonged by FLU at doses of 1.0 and 16.0 mg/kg（P<0.05）.In contrast,LORR induced by ZOL（50 mg/kg）was effectively antagonized by FLU.FLU dose dependently increased the latency(F_{（5,53）}=9.82,P<0.01)and reduced the duration(F_{（5,53）}=11.06,P<0.01)of LORR induced by ZOL.In addition,percentage of ZOL-induced LORR was reduced to 50%by FLU at dose of 1.0 mg/kg.Conclusions:α₁β₂ receptor showed different sensitivity to BZDs with distinct chemical structures,bothαandβsubunits contribute to the sensitivity ofαβreceptor to DZP,indicating that non-classical binding sites depended on certain receptor subtypes and benzodiazepine structures.In addition,non-classical BZD effects on distinct GABA_Areceptor subtypes（αβorαβγ）were different.Effects of DZP on some cortical neurons could not be fully antagonized by flumazenil,however,the flumazenil-insensitive effects were not observed on hippocampal neurons,indicating that non-classical benzodiazepine binding sites may exist in neuronal GABA_A receptors.Both the classical and non-classical binding sites were involved in BZDs-induced anesthesia in mice.The classical binding sites contribute mainly to induction of anesthesia,whereas non-classical binding sites seem to be more related to maintance of anesthesia.Difference in the interaction of DZP,MDZ and ZOL with classical and non-classical binding sites results in their distinct sensitivity to FLU in the LORR behavior.